Chronic diarrhea is a curse for AIDS patients through its association with advanced lymphopenia and mortality. Its incidence is greatest in sub-Saharan Africa where it may undermine absorption of HAART. Diagnosis is flawed by the poor sensitivity of available tests for pathogen detection. We hypothesize that diagnostic failure to detect opportunistic infection accounts for a large proportion of AIDS diarrhea cases given "no diagnosis." In this proposal we will develop a sensitive bead-PCR assay that detects multiple pathogens in single reaction and then test our hypothesis on 401 existing stool specimens from our prior AIDS cohort study from Tanzania, whereby 78% of diarrhea cases revealed no pathogen via microscopy or antigen detection. Based on the practicality of an R21 application, our experience with protozoa, and reviewer's comments, we will limit this project to Cryptosporidium, Giardia, Isospora, and the two major intestinal Microsporidia. The assay involves conventional PCR using biotinylated primers followed by hybridization of amplicon to oligonucleotide probes that are covalently linked to uniquely colored beads. Fluorescence is generated by addition of streptavidin-PE and quantitated per bead color on a Luminex suspension array machine, such that up to 100 pathogen species can be simultaneously resolved. Development of the assay is not trivial and requires attention to the initial multiplex PCR and optimization of amplicon-probe signal-to-noise ratio. We have preliminary data that the procedure is sensitive and specific, as well as the necessary reagents (parasite DNA), equipment (Luminex machine), and real-time PCR assays for Giardia and Cryptosporidium that can be used to validate the new procedure. Through generation of preliminary data from this proposal we will plan to transfer the technique to our ongoing clinical site in Tanzania, where it can be used on a research basis to characterize prevailing pathogens in order to devise appropriate management algorithms. Finally, syndrome-based multiplex diagnosis is the platform of the future for clinical microbiology, and if this assay works on stool it should work on sputum, blood, or spinal fluid. [unreadable] [unreadable] [unreadable]